Dimethanesulfonates of certain 7-(o-aminomethylphenylacetamido)-3-((heterocyclylthio)methyl)-ceph-3-em-4-carboxylic acids

ABSTRACT

1. A COMPOUND OF THE FORMULA   2-(NA-OOC-),3-(R-S-CH2-),7-((NA-O3S-CH2)2-N-CH2-(1,2-   PHENYLENE)-CH2-CO-NH-)-2-CEPHEM   WHEREIN R IS   1-(H3C-)-TETRAZOL-5-YL-, -(5-(H3C-)-1,3,4-THIADIAZOL-2,3-   YLENE)-, OR 1H-1,2,3-TRIAZOL-4-Y

United States Patent 3,840,535 DIMETHANESULFONATES OF CERTAIN 7-(0- AMINOMETHYLPHENYLACETAMIDO) 3- [(HETEROCYCLYLTHIO) METHYL]-CEPH-3- EM-4-CARBOXYLIC ACIDS Murray Arthur Kaplan, Syracuse, and Alphonse Peter Granatek, Baldwinsville, N.Y., assignors to Bristol- Myers Company, New York, N.Y. No Drawing. Filed Aug. 23, 1972, Ser. No. 283,121 Int. Cl. C07d 99/24 US. Cl. 260-243 C 4 Claims ABSTRACT OF THE DISCLOSURE 7 (o aminomethylphenylacetamido)-3-(l-methyl-S- tetrazolylthio)methyl]ceph-3-em-4-carboxylic acid, 7-(0- aminomethylphenylacetamido)-3-[S-methyl 1,3,4 thiadiazolylthio)methyl]-ceph-3-ern-4-carboxylic acid and 7 (o aminomethylphenylacetamido-3-[S-( 1,2,3 triazole- 5-yl)-thiomethyl]-ceph-3-em-4-carhoxylic acid are valuable as antibacterial agents in animals, including man, and areespecially useful in the treatment of infectious diseases caused by many Gram-positive and Gram-negative bacteria. They are administered by injection and for this purpose the preferred formulation, as usual, is a true solution in water. Ordinary methods of converting these water-insoluble zwitterions to a water-soluble salt, such as the sodium salt, require a pH so high that the product is inactivated rapidly and thus cannot be used to formulate an injectable product even when the water is to be added just before use. The present invention solves this problem by converting these cephalosporins to their dimethane-sulfonate sodium salts (containing three atoms of sodium) as by reaction with sodium formaldehyde bisulfite in the presence of sodium 2-ethylhexanoate.

BACKGROUND OF THE INVENTION (1) Field of the invention The cephalosporin derivatives of the present invention possess the usual attributes of such compounds and are particularly useful in the treatment of bacterial infections by injection.

(2) Description of the prior art The three cephalosporins named,

7- (o-aminome thylphenylacetamido -3-( l-methyl-5-tetrazolylthio) methyl] ceph-3-em-4-carboxylic acid,

7- oaminomethylphenylacetamido) -3- (5 methyl- 1 3 ,4-

thiadiazolylthio) methyl] -ceph-3 -em-4-carboxylic acid and 7- (o-aminomethylphenylacetamido-3- [S- 1,2,3-triazole- 5-yl-thiomethyl] ceph3-em-4-carb oxylic acid are disclosed by R. U. Lemieux and R. Raap in US. application Ser. No. 142,337 filed May 11, 1971 and issued Oct. 16, 1973 as US. 3,766,175. That application also contains a review of the prior art in the field of semisynthetic cephalosporins.

The literature contains various disclosures of methanesulfonates of antibiotics. Thus Belgium 742,728 (Farmdoc 41466R) discloses the mono-methanesulfonate of ampicillin. U.S. Pats. 3,268,508 and 3,295,246 disclose the conversion of one or more of the four free amino groups of kanamycin to the methanesulfonate derivative. Sodium bacitracin methanesulfonate is disclosed in US. 3,205,- 137 and for neomycin see I. Antibiotics (Japan) 12, 114- 115 (1959). The injectable antibiotic colistimethate (marketed by Warner-Chilcott as Coly-Mycin) is the sodium salt of the methanesulfonate of colistin; Merck Index cites Koyama et al., Japan 4898 (1957). Colistimethate is reviewed in detail on pages 315-320 of the US. Dispensatory 26th Edition, I. B. Lippincott Comice pany, Philadelphia, Pa. It is described as the pentasodium salt of the penta(methanesulfonic acid) derivative of Colistin A at pages 621-622 of U.S.P. XVII. US. Pat. 2,599,950 in Example VIII discloses Polymyxinform aldehyde-sodium bisulfite compound. See also UK. 874,- 028, 902,992 and 896,774 and Japan 15,948/61.

SUMMARY OF THE INVENTION 7 (0 aminomethylphenylacetamido)-3-(l-methyl-S- tetrazolylthio)methyl]ceph 3 em-4-carboxylic acid, 7- (o-aminomethylphenylacetamido) 3-[(5 methyl-1,3,4- thiadiazolylthio)methyl]-ceph-3-em-4-carboxylic acid and 7 (0 aminomethylphenylacetamido-3-[S-(l,2,3-triazole- 5-yl)-thiomethyl]-ceph-3-em-4-carboxylic acid are valuable as antibacterial agents in animals, including man, and are especially useful in the treatment of infectious diseases caused by many Gram-positive and Gram-negative bacteria. They are administered by injection and for this purpose the preferred formulation, as usual, is a true solution in water. Ordinary methods of converting these water-insoluble zwitterions to a water-soluble salt, such as the sodium salt, require a pH so high that the product is inactivated rapidly and thus cannot be used to formulate an injectable product even when the water is to be added just before use. The present invention solves this problem by converting these cephalosporins to their dimethane-sulfonate sodium salts (containing three atomsv of sodium) as by reaction with sodium formaldehyde bi- OONa wherein R is The preferred embodiment of the present invention is the compound of the formula There is further provided by the present invention the process for the production of wherein R is ii N/ w or which comprises mixing a zwitterion of the formula wherein R is with at least 2.0 moles of sodium formaldehyde bisulfite per mole of zwitterion, water and at least 2.0 moles of a strong sodium base, e.g. sodium 2-ethylhexanoate in acetone or isopropyl alcohol and warming the mixture above room temperature, e.g. above 40 C. and preferably in the range of 40-45 C., for a brief period of time until there is produced a solution of the desired product. The product is recovered as a solid by precipitation, e.g. by the addition of a water-soluble, substantially anhydrous solvent which is preferably an alcohol such as ethanol or isopropanol.

The use of lesser amounts of sodiumformaldehyde bisulfite produces a mixture of unreacted starting material and di-methanesulfonate of the present invention; it was unexpected to find that mono-methanesulfonate was not then produced.

In the treatment of bacterial infections in man, the compounds of this invention are administered parenterally, in accordance with conventional procedures for antibiotic administration, in an amount of from about 5 to 200 mg./kg./day and preferably about 5 to 20 mg./kg./ day in divided dosage, e.g., three to four times a day. They are administered in dosage units containing, for example, 125, 250 or 500 mg. of active ingredient alone or, if desired, with suitable physiologically acceptable carriers or excipients or other agents such as preservatives (e.g. parabens) or local anesthetics (e.g. procaine or lidocaine). The dosage units are in the form of liquid preparations and preferably as true solutions in water. The preferred dosage unit is a one-dose vial containing only a sterile amount, e.g. 125 mgm., of a solid compound of this invention. Just prior to use 1 ml. or 2 ml. of sterile water is added to form a solution of the product which is then injected.

DESCRIPTION or THE PREFERRE methyl 5 tetrazolylthio)methyl]ceph 3 em v 4 wherein R represents N 1.1.. .1. C2115. N L- HOCHISOQNa CHa(CH );iJH-COONa N (30% SEE in acetone) 7 1. Heat; inwater I 1 I 12. Carbon treat and filter 3. Add filtrate to anhyd rous alcohol a onzmonasoznor v p, i"

SOudQ-CHF-ii -NH-Qnfi cin 1 r cir.-s 1i 0- wherein R represents r U N-CI-Ia Hlo E A Procedure: Put 2.0 moles (abo 1952 011 an anhy-" drous basis) of 7-(0 aminomethylphenylacetamido-3-[(l methyl-S-tetrazolylthio)methyl]ceph-3-em 4 carboxylicjf acid, 540 g. of sodium formaldehyde bisulfite (2.031 moles), 3000 ml. of water and 2700 ml. (4.87 moles) of; 30% SEH (sodium 2-ethylhexanoate) in acetone in' a suitable tank and with stirring heat the mixture to 45 C. The mixture dissolves in about 10 min. to a yellow solution.

After 15 minutes of heating add 50 g. of decolorizingv charcoal (Darco KB) to the solution and stir '15 I'nin. more at 40-45 C.

Filter through diatomaceous earth ("Dioalite) ,after' heating the reaction at 40-45 C. for atotalof 30 minute's. If

Wash the carbon cake with 2000 m1. of 50% ethanolwater.

Combine the filtrates, adjust'to 25 C. and; add the solution at 25 C. to 112 liters of rapidly stirred ethyl alcohol. A fine white amorphous precipitate of the di(sodium-methane sulfonate) of sodium ,7-(b-aminbmethylphenylacetamido 3 1 methyl 5 tetra'zolylthio)methyl]ceph-3-em-4-carboxylate form's.

Stir the suspension for about 10 minutes and then filter and wash the cake with 15 liters of 100%, ethyl alcohol.

Dry the cake at 50-55 C. in an oven with air circulating for about 2 hours and then under vacuum at-46"; mm. for24hrs. H

The yield is about 1200-1400 g. of amorphous, white; solid di(sodium-methane sulfonate') of sodium 7- (oaminomethylphenylacetamido 3 [(1 methyl 5 tetrazolylthio) methyl] ceph-3-em-4-carboxylate. The product usually contains several percent water and possibly a trace of ethanol. 1 a

. Example2, I The following slurry is prepared. 2.19 grams of sodium-formaldehyde bisulfite (2*equivalents). 1 3.5 g. of 7 (o aminomethylphenylacetamido 45- ['(1- 4 wear:

boxylic acid zwitterion (100-200 mesh).-

25 m1. of water (volume can be varied). -14 ml. of 30% SEH-isopropa'nol. I A near solution is obtained in about 0.5 hours.:of rapid:

stirring at 24" C. The temperature of'the mixture is raised rapidlyto 40-43 C. This is maintained 'for about'two minutes and then rapidly cooled to;-20-23 C. i; i

The solution is filtered to remove some insolubles (total time in solution should not exceed two hours). The pH 7.3 solution is'added-overa 5 'in'inuteperiod' to 600 ml. of very rapidly stirring absolute "ethanol (b'therfi alcohols such as anhydrous isop'ropanol may be used). An amorphous precipitate of the di(sodium-methanesulfonate) of sodium 7-(0-aminomethylphenylacetamido)-3- [(1 methyl 5 tetrazolylthio)methyl]ceph 3 em 4- carboxylate forms. The mixture is stirred for 5 minutes. The precipitate is collected by filtration, Washed with 60 ml. of ethanol (or isopropanol) and vacuum dried at 50 C. for 24 hours. The yield is about 4.3 g.

The product is soluble in water at about pH 7 to the extent of at least 200 mgm./ml. Such a solution is stable for at least two hours at room'temperature; more dilute solutions are stable even longer. The product shows the same antibacterial spectrum as the parent zwitterion and is fully biologically active whether it has hydrolyzed back to the zwitterion or not.

Example 3 The following slurry is prepared:

2.19 grams of sodium-formaldehyde bisulfite (2 equivalents).

3.5 g. of 7 aminomethylphenylacetamido)-3-[(5- methyl 1,3,4 thiadiazolylthio)methyl]ceph 3 em 4- carboxylic acid zwitterion (100-200 mesh).

25 ml. of water (volume can be varied).

14 ml. of 30% SEH-isopropanol.

A near solution is obtained in about 0.5 hour of rapid stirring at 24 C. The temperature of the mixture is raised rapidly to 40-43 C. This is maintained for about two minutes and then rapidly cooled to 20-23" C.

The solution is filtered to remove some insolubles (total time in solution should not exceed two hours).

The pH 7.3 solution is added over a 5 minute period to 600 ml. of very rapidly stirring-absolute ethanol (other alcohols such as anhydrous isopropanol may be used). An amorphous precipitate of the di(sodium-methanesulfonate) of sodium 7 (0 aminomethylphenylacetamido-3- [(5 methyl 1,3,4 thiadiazolylthio)methyl]ceph 3- em-4-carboxylate forms. The mixture is stirred for 5 minutes. The precipitate is collected by filtration, washed with 60 ml. of ethanol (or isopropanol) and vacuum dried at 50 C. for 24 hours. The yield is about 4.3 g.

The product is soluble in water at about pH 7 to the extent of at least 200 mgm./ml. Such a solution is stable for at least two hours at room temperature; more dilute solutions are stable even longer. The product shows the same antibacterial spectrum as the parent zwitterion and is fully biologically active whether it has hydrolyzed back to the zwitterion or not.

Example 4 The following slurry is prepared:

2.19 grams of sodium-formaldehyde bisulfite (Z-equivalents).

3.5 g. of 7-(o-aminomethylphenylacetamido-3-[S-(1,2, 3-triazole 5 yl)thiomethyl]ceph-3-em-4-carboxylic acid zwitterion (100-200 mesh).

25 ml. of water (volume can be varied).

14 ml. of 30% SEH-isopropanol.

A near solution is obtained in about 0.5 hour of rapid stirring at 24 C. The temperature of the mixture is raised rapidly to 4043 C. This is maintained for about tw o minutes and then rapidly cooled to 20-23 C.

The solution is filtered to remove some insolubles (total time in solution should not exceed two hours).

The pH 7.3 solution is added over. a 5 minute period to 600 m1. of very rapidly stirring absolute ethanol (other alcohols such as anhydrous isopropanol may be used). An amorphous precipititate off the di(sodium-,methanesulfo nate) of sodium 7-.(01aminomethylphenylacetamido)-3- [S (1,2,3 triazole 5 yl)thi0methy1]ceph 3 em 4- carboxylate forms. The mixtureis stirred for 5 minutes. The precipitate is' collected by filtration, washed with 60 ml. of ethanol (or isopropanol) and vacuu'm dried at 50 C. for 24 hours. The yield is about 4.3 g. t

The product is soluble in water at about pH 7 to the extent of at least 200 mgm./ml. Such a solution is stable for at least two hours at room temperature; more dilute solutions are stable even longer. The product shows the same antibacterial spectrum as the parent zwitterion and is fully biologically active whether it has hydrolyzed back to the zwitterion or not.

Starting Materials NB S CHzC 020113 -v 1. NaNa CHzC 03 C H3 2.7ldrol. 0mm

H Pd-C 01120 0 11 CHzNs B O C-Na C H: C 02H -v CHQNHg 2,4-DNP -C H20 0211 D C C CHzNHB O C CHzNH-B O C Methyl o-bromomethylphenylacetate: A mixture of methyl o-methylphenylacetate (82.0 g., 0.50 mole), N- bromosuccinimide (89.0 g., 0.50 mole), benzoyl peroxide (1.0 g.) and carbon tetrachloride (800 ml.) was heated under reflux for 2 h. while irradiated with a 750 watt light source. The succinimide was removed by filtration, the solvent removed from the filtrate and the residue distilled in vacuo to give 90.1 g. (74%) of product, b.p. 105 (0.4 mm.); N.M.R. (CCI singlets at r 2.85 (4H), 5.50 (2H), 6.31 (2H) and 6.38 (3H).

o-Azidomethylphenylacetic acid: A mixture of methyl o-bromomethylphenylacetate (90.1 g., 0.371 mole), sodium azide (26.0 g., 0.40 mole) and 10% aqueous acetone (750 ml.) was stirred at room temperature for 3 h. The solvent was removed under reduced pressure and the residue treated with ether (300 ml.) and water ml.). The crude methyl o-azidomethylphenylacetate (74.8 g.), obtained after drying and concentrating the ether solution, was dissolved in ml. of methanol. This solution was cooled in ice and treated with 150 ml. 3 N methanolic sodium hydroxide. The mixture was left at room temperature for 1 h., then concentrated to dryness and the residue dissolved in water. The aqueous solution was acidified, the product collected by filtration, dried and recrystallized from ethyl acetate-n-hexane to give 49.5 g. (70%) of the acid, mp. 116-118; N.M.R. (CD01 sharp singlets at 'r 2.75 (4H), 5.63 (2H) and 6.28 (2H);

1 22i? 2100 and 1700 cmf Anal.Calcd. for C H N O C, 56.53; H, 4.75; N, 21.98. Found: C, 56.37; H, 4.65; N, 21.74.

oAminomethylphenylacetic acid: A mixture of o-azidomethylphenylacetic acid (9.6 g., 0.050 mole), 10% Pd on charcoal (2.5 g.), methanol (150 ml.) and 1 N hydrochloric acid (50 ml.) was hydrogenated at 30 p.s.i. for 3.5 h. The mixture was filtered, concentrated under reduced pressure to a volume of approximately 30 ml. and extracted with ether. From the ether extract 1-2 g. of impure starting material was recovered. The aqueous solution was adjusted to pH 5.0 with dilute ammonium hydroxide and cooled in ice. The white solid precipitate was collected by filtration, washed successively with ice-water,

methanol and ether, and dried in vacuo over P Yield 5.4 g. (65%), m.p. 179- 181 (decomp.); N.M.R. (CF CO H): 'r 2.54 (s, 4H) 5.48 (q, 2H) and 6.00 (s, 2H).

o-Tert butoxycarbonylaminomethylphenylacetic acid: Triethylamine (14.4 g., 0.143 mole) Was added to an ice-cooled suspension of o-aminomethylphenylacetic acid (10.3 g., 0.0624 mole) in 100 ml. of water followed by the addition of a solution of tert.butoxycarbonyl azide (11.4 g., 0.080 mole) in 75 ml. of THF. The reaction mixture was stirred at room temperature for 16 h., then most of the THF was removed under reduced pressure. The aqueous solution was Washed with ether, layered with 125 ml. of ethyl acetate and with ice-cooling brought to pH 3.5 with dilute hydrochloric acid. The ethyl acetate solution was dried, concentrated and the solid residue recrystallized from ethyl acetate-n-hexane (1:1) to give 14.4 g. (87%) of white needles m.p. 114-116.

Anal.-Calcd. for C H NO C, 63.39; H, 7.22; N, 5.28. Found: C, 63.44; H, 7.21; N, 5.42.

2,4-dinitrophenyl o-te rt. butoxycarbonylaminophenylacetate: -N,N-dicyclohexylcarbodimide (1.0 g., 0.0050 mole) was added to an ice-cooled solution of 0-tert.- butoxycarbonylaminomethylphenylacetic acid (1.33 g., 0.0050 mole) and 2,4-dinitrophenol (0.92 g., 0.0050 mole) in 12 ml. of anhydrous tetrahydrofuran. The reaction mixture was kept at room temperature for one hour, then the precipitated N,N'-dicyclohexylurea was removed by filtration. The solvent was removed from the filtrate to give the activated ester as a viscous yellow oil.

o-Tert. butoxycarbonylaminomethylphenylacetic acid can be prepared in quantitative yield from tert.-butoxycarbonyl azide and the amino acid by using triethylamine as the base.

The BOG-amino acid reacts with thionyl chloride in the presence of triethylamine (methylene chloride as solvent) or pyridine (benzene as solvent) to give the BOC-amino acyl chloride which can be directly coupled with the compound of the formula doz nta N11 wherein R has the meaning set out above in methylene chloride solution in the presence of triethylamine. The protecting group can subsequently be removed by treatment with cold trifluoroacetic acid.

Exactly 200 g. of 7 aminocephalosporanic acid (7-ACA) Was suspended in 500 ml. of acetone and a solution of 240 g. of p-toluenesulfonic acid in 500 ml. of acetone was added in one charge. After stirring for five minutes, at room temperature, the mixture was filtered through diatomaceous earth (Super Gel) and the bed washed with 150 ml. of acetone (the insoluble matter weighed about 30 g.). Then 80 ml. of water was added to the filtrate and, while stirring, the p-toluene-sulfonate salt crystallized out after scratching on the inside of the flask with a glass rod. The suspension was stirred in an ice-salt bath for thirty minutes and filtered cold. It was washed with 2X 200 ml. of cold acetone (0 C.) and air dried; yield 250 g. of salt. This p-toluene-sulfonate salt of 7-ACA was stirred in 2 liters of methanol and the insoluble matter filtered through Super Cel. The filtrate was placed in a five liter 3 neck flask and 2 liters of water were added. Then the pH was adjusted to 4 by the addition of concentrated ammonium hydroxide with cooling and the suspension stirred for one hour at 0 C. The product was collected by filtration and washed with 2x 100 ml. H O (0 C.) and3 1 liter acetone (room temperature). Aft er air drying, the yield of 7-ACA was 145 g. I

Reference: Glaxo, British Pat. 1,104,938 (1968).

Z-mercapto-S-methyl-1,3,4-thiadiazole: Lit. ref. US. Pat. 3,516,997 (1970); J. Antibiotics, 23, 131-36 (1970).

11.5 g. (0.1 mole) of 2-arnino-5-methyl-1,3,4thiadiazole was carefully ground together with 32 g. (0.45 mole). of sodium nitrite and slowly added to 160ml. of 48% HBr containing 50 mg. of powdered copper at 10 C. with stirring. After the addition was completed, the solution was stirred at 5 C. for one hour and then at 20 C. for one and a half hours. The pH was adjusted to 9.5 by addition of 50% KOH and the solution was heated to 60 C. At 60 C. the pH was readjusted to 9.5 by addition 50% KOH. The solution was cooled and filtered. The precipitate was dissolved in ether and the filtrate was extracted with 2x 200 ml. ether. The combined ether solutions were dried over sodium sulfate and evaporated to dryness. The product was recrystallized from benzene Skellysolve B. Yield 12 g., m.p. l05107 C.

12 g. (.07 mole) of 2-bromo-5-methyl-1,3,4-thiadiazole and 5 g. (.07 mole) of thiourea were dissolved'in-40 ml. of ethanol and refluxed for one and a half hours on a steam bath. This solution was added to 4.5 g. (.08 mole) of KOH in 65 ml. H 0 and the mixture heated to boiling for five minutes. The ethanol was removed under vacuum and the pHof the aqueous solution adjusted to 3 by addition of 3 N HCl. The product crystallized out and after cooling at 0 C. for one hour was collected by filtration, washed with cold water and recrystallized from 100% ethanol. Yield 5 g., m.p. 186-187 C.

Analysis.Calcd. for C H N S C, 27.25;-H, 3.05; N, 21.19; S, 48.51. Found: C, 27.20; H, 3.34; N, 21.18; S, 48.48. i

7 amino-3-(S-methyl-1,3,4-thiadiazol-2-ylthiomethyl)- 3-cephem-4-carboxylic acid (IIa): To a stirred suspension of 2.72 g. (0.01 mole) of 7-ACA in 50 ml. of 0.1- M, pH 6.4 phosphate bufler, was added 1.68 g. (0.02 mole) of NaHCO followed by 1.45 g. (0.011 mole) of Z-mercapto-S-methyl-1,3,4-thiadiazole and the mixture heated and stirred at 60 C. for five hours. The resulting slurry was then allowed to cool to about 22 C. over a one hour period. The crystalline precipitate was collected by filtration, Washed with water and air dried. Yield 1.3 g., dec. pt. 206 C. Scaling up the reaction .10 gave 18.0 g. I

Analysis.--Calcd. for C H N O S C, 38.37; H, 3.52; S, 27.96. Found: C, 39.06; H, 3.91; S, 26.67.

Synthesis of potassium 1,2,3-triazole-5-thiolate mmoles). When the addition was completed, the mixture-v was stirred for 1 hour at 0, the solid was collected by filtration and driediwvacuo. The melting point of the.

crude material (23.3 g.) thus obtained wasobserved somewhere in the region 232 to 257. Goerdler reported,m.p.

267 for the pure material. A small second crop. (2.1 g.)

was obtained by evaporation of the mother liquor in v'qciio.v

The total yield was therefore 40%,

1,2,3-triazole-5-thiol: A solution of the above benzamido compound (8.2 g., 40 mmoles) in 2N sodium hydroxide (80 ml., 160 mmoles) was heated under reflux temperature in a nitrogen atmosphere for 24 hours. The solution was cooled to in ice, and concentrated hydrochloric acid (26 ml.) was added, while a continuous stream of nitrogen was passed through the solution. The benzoic acid which precipitated was collected by filtration; the filtrate was saturated with sodium chloride andthe additional benzoic acid which separated was removed by filtration. The filtrate was immediately extracted with ethyl acetate, the extract was washed with saturated salt solution, dried over magnesium sulfate and then evaporated in vacuo. The viscous oil which remained was immediately evaporatively distilled in vacuo (70-75/0.001 mm.) to give an oil (2.84 g., 70%) which solidified (m.p. 52-59"; Goerdler reported m.p. 60) spontaneously.

Potassium 1,2,3-triaz0le-5-thiolate: To a solution of the above thiol (2.84 g., 28.1 mmoles) in absolute ethanol (28 ml.) was added 1.93 N alcoholic potassium hydroxide solution (14.5 ml.). The solution was then diluted with anhydrous ether until crystallization of the salt was completed. The solid was collected by filtration, washed with ether, and dried in vacuo. The salt obtained in this manner (3.65 g., 93%) had m.p. 225 with decomposition.

It is important to note that the conversion of the benzamido thiadiazole to the triazole thiol is known to proceed via 5-amino-1,2,3-thiadiazole [G. Goerdler and G. Gnad, Chem. Ber. 99, 1618 (1966)].

N-- s t it a l IT 5-amino-1,2,3-thiadiazole can be prepared by an alternative route, not involving diazornethane [D. L. Pain and R. Slack, J. Chem. Soc. 5166 (1965)].

Alternate Synthesis of 1,2,3-Triazole-5-Thiol Preparation of the starting materials:

O. Diels, Chem. Ber. 47, 2183 (1914).

CHzCHKOC Hs):

l HCOOH/HCI N CHICH(OH)1 D. L. Pain and R. Slack, J. Chem. Soc. 5166 (1965). G. Goerdeler and G. Gnad. Chem. Ber. 99, 1618 (1966).

Ethyl Carbazate II II HQOZOCOCZHB NzHrHzO OzNNHCOCzHa 0. Diels. Chem. Ber. 47, 2183 (1914).

Hydrazine hydrate 44 g.) was added with vigorous stirring over a 10 min. period to diethyl carbonate (100 g.). After a short induction period, the two phase system began to warm up and eventually a clear single phase resulted (ca. 10 min.) The reaction temperature did not exceed 50. Stirring was continued until a single phase resulted and then the clear solution was set aside for 7 hours (Note 1). The solution was then fractionated in vacuo to give a clear liquid b.p. 93.5- 95.5/10 mm. The distillate (70.1 g., 78.7%) crystallized spontaneously on standing.

Note 1. A reaction which was left at room temperature for 16 hours gave precisely the same yield of product.

1 1 Phthalimidoacetaldehyde Hydrate NCHzC H(O CzH)2 7 262.29 HCOOH/HCI /NCH2 C H O H): O

W. Siedel and H. Nahm. Ger. 928,711 (1955); Chem. Abstr. 52, 5471 (1958).

.A mixture of dry potassium phthalimide (92.6 g., 500 mmoles) and acetamide (120 g.) was placed in an oil bath maintained at ca. 150 When the acetamide had melted and stirring became possible (Note 1), bromoacetaldehyde diethyl acetal (98.5 g., 500 mmoles) was added to the stirred mixture over a 2 hour period. When the addition was completed, the mixture was stirred at 140 (reaction temperature for 1 hour), then the oil bath temperature was raised to ca. 180 (reaction temperature 170). The reaction temperature was held at 170 for /2 hour, the hot mixture was then poured with vigorous stirring into a large volume of ice-water, the solid was collected by filtration, it was washed well with water, and then sucked as dry as possible. This solid was then stirred vigorously with dichloromethane (600 ml.), the insoluble solid was filtered off, the filtrate was dried over sodium sulfate, treated with a few grams of activated charcoal, filtered, and evaporated in vacuo. The residue was broken up, shaken with water and collected by filtration. The solid was pulverized as much as possible while still in the Biichner funnel, it was washed well with water and then dried in vacuo. The crude product, m.p. 6070 (101.8 g., 77.5%), was used in the next step without purification.

The above acetal (90 g.), 85% formic acid (90 ml.) and concentrated hydrochloric acid (9 ml.), were heated together on an eflicient steam bath for minutes (Note 2). The hot solution was diluted with water (810 ml.) and the evaporated in vacuo (water pump, Note 3) to a volume of about 150 ml. The mixture was cooled in ice, the solid was collected by filtration, washed with icecold water, and then dried in vacuo. The white solid thus obtained (65.7 g., 92.4%) melted at ca. 90, resolidified (Note 4), and then melted again at 106-109. Evaporation of the filtrate and washings as above gave a further 1.1 g. of a solid m.p. 107-158. It was advisable to recrystallize this material from a large volume of petroleum ether (b.p. 90-120) containing a trace of benzene. There was thus obtained a further 0.66 g. of product with m.p. 106-109 (Note 5).

Notes 1. It was advisable to stir the mixture mechanically.

2. Shorter heating periods resulted in the recovery of starting material.

3. Higher temperatures gave more of the high melting material described in the text.

4. -If, the melt did not resolidify spontaneously at this stage, the capillary was removed from the melting point apparatus. When the sample had crystallized, it was returned to the apparatus; it then had m.p. 106-109. This material could be used without purification.

. .5. The authors (Siedel and Nahm) reported a melting point of 129 for the hydrate. The material with m.p. ca. then 106109 described herein, contains the hydrate as indicated by medium intensity absorptions at 3400 cm.- The recrystallized material, m.p. 106l09, is evidently the pure aldehyde. This aldehyde is reported (R. J. Collins, B. Ellis, S. B. Hansen, H. S. Mackenzie, R. J. Moualim, V. Petrow, O. Stephenson, and B. Sturgeon. J. Pharm. and Pharmacol, 4, 693 (1952)) to have m.p. 112.

Phthalimido Acetaldehyde Ethoxycarbonyl Hydrazone N CH. CH=NNH C 02 ca t D. L. Pain and R. Slack. J. Chem. Soc. 5166 (1965).

A solution of ethyl carbazate (38.9 g., 370 mmoles) in warm toluene (300 ml.) was added dropwise to a magnetically stirred solution of the aldehyde hydrate 65.7 g., 317 mmoles) in toluene (600 ml.), containing 3 small crystals of toluene sulfonic acid hydrate, over a 15 minute period. The apparatus was maintained in a water bath at 75 during the addition. When the addition was completed, stirring was continued at 75 (bath temperature) for 3 hours, the mixture was then cooled to room temperature, the product was collected by filtration, and dried in vacuo. The white solid (71.0 g., 81.2%) thus obtained had m.p. 163 (prior softening), and could be used without further purification. The authors reported that recrystallized material (ethanol) had m.p. 168-169.

5-Phthallmid0-1,2,3 Thiadiazole NCHzCH=N-NHCOzCzH5 $001: v

D. L. Pain and R. Slack. J. Chem. Soc. 5166 (196 5).

Thionyl chloride (66 ml.) was cooled to ca. 5 and the hydrazone (60 g., 217 mmoles) was added all at once. The cooling bath was removed, and in a short time a vigorous reaction accompanied by a copius evolution of gases ensued. When this subsided, the mixture was set aside at room temperature for 16 hours (the mixture was swirled occasionally to ensure good contact of the re agents). Benzene (300 ml.) was added to the mixture and the whole was evaporated to dryness in vacuo. A

further quantity of benzene was added to the residue and the mixture was evaporated to dryness once more. It was usually necessary to repeat this process one more time to completely remove occluded hydrogen chloride. The solid thus obtained was slurried with benzene, collected by filtration, and dried in vacuo. It weighed 40.3 g. (80.4%) and had m.p. 21822l dec. This material was pure enough to use in the next step, but recrystallization could be accomplished from a large volume of alcohol. It then had m.p. 222-224 dec. The authors reported m.p. 225-226 dec. for the purified material.

-Amino-1,2,3-Thiadiazole D. L. Pain and R. Slack, J. Chem. Soc. 5166 (1965).

A solution of hydrazine hydrate (100%, 10.5 g.) in absolute ethanol (150 ml.) was added dropwise to a refluxing, mechanically stirred, mixture of the phthalimido compound (23.1 g., 100 mmoles) in absolute ethanol (320 ml.) .over a /2 hour period. When the addition was completed, the mixture was stirred under reflux for an additional /2 hour. The mixture was cooled to room temperature, the solid phthalhydrazide was removed by filtration, and the filtrate was concentrated in vacuo to a volume of ca. 70 ml. The precipitated phthalhydrazide was removed by filtration, washed with a little alcohol, and the combined filtrate and washings were evaporated to dryness in vdcuo. The residual solid (8.110.1 g., 80100%) was sufllciently pure to be used in the next step. It could, however, be recrystallized from a small volume of alcohol or from a benzene alcohol mixture (3:1) to give a solid m.p. 143145 dec. The recovery was not good and several additional crops could be obtained by working up the mother liquor. The authors reported m.p. l45147 for this compound.

1,2,3-Triaz0le-5-Thio1 I. Goerdeler and G. Gnad. Chem. Ber. 99, 1618 (1966).

A solution of S-amino-l,2,3-thiadiazole (4.04 g., 40 mmoles) in 2 N sodium hydroxide (40 ml.) was boiled under reflux in a nitrogen atmosphere for 5 minutes. The solution was cooled in ice (nitrogen was bubbled through the solution throughout the workup period), acidified with hydrochloric acid to pH 1 (indicator paper), saturated with salt, and then extracted as rapidly as possible with ethyl acetate. The extract was dried briefly over magnesium sulfate (nitrogen was bubbled through the extract) and then evaporated in vacuo. The residual oil was immediately (Note 1) subjected to evaporative distillation (80/ 0.001 mm.) to give a clear distillate which crystallized spontaneously. The yield of the pure thiol varied between 64 and 84% depending on the purity of the starting thiadiazole.

Note 1. In the crude state, the thiol is very sensitive to aerial oxidation to the disulfide. The purified thiol possesses considerably more stability and can be kept at 0 in stoppered vessels for several weeks without significant change.

7-amino-3-[S-(1,2,3-triazole-5 yl) thiomethyl] 3- cephern-4-carboxylic acid: Ten grams (0.075 mole) of 5- mercapto-l,2,3-triazole potassium salt was added to a stirred slurry of 19 g. (0.07 mole) of purified 7-aminocephalosporanic acid and 5.9 g. (0.07 mole) of NaHCO in 350 ml. of 0.1M phosphate butter (pH 6.4) and the mixture heated and stirred at 55 C. for 3 /2 hours under a nitrogen atmosphere. The resulting solution was cooled to 22 C. and the pH adjusted to 5.5 with 40% H PO The resulting precipitate was filtered off, washed with cold water (50 ml.) and air dried. The yield of 7-amino- 3-[S-(1,2,3-triazole-5-yl)-thiomethyl]-3-cephem 4 carboxylic acid was 8 g., dec. pt. 230 C. Ir analysis showed some decomposition of the fl-lactam ring but it was used as is for the next step.

Anal.-Calcd. for C I-I N O S C, 38.39; H, 3.54. Found: C, 38.36; H, 3.78.

7-amino-3-(2-methyl-1,3,4-thiadiazole-5 thiornethyl)- A -cephem-4-carboxylic acid: As disclosed in South Africa 70/2,290, to a stirred mixture of 27.2 g. (0.1 mole) of 7- aminocephalosporanic acid in 200 ml. of water and 100 ml. of acetone was added a saturated solution of sodium bicarbonate to a pH of 7.9. This solution was placed in an C. bath and when the internal temperature had reached 45 C. a solution of 19.6 g. (1.15 mole) of 2-methyl-1,3,'4- thiadiazole-S-thiol in 200 ml. of acetone was added. The mixture was heated in the 80 C. bath for threehours and then cooled in 10 C. and the pH was adjusted to 3.9 by the addition of 6 N hydrochloric acid. The cold mixture was stirred for 15 minutes and the solid was collected, washed with acetone, and dried. There was obtained 24 g. (70 percent) of 7-amino-3-(Z-methyl-1,3,4-thiadiazole-5- thiomethyl)-A -cephem-4-carboxylic acid.

7-amino-3- l-methyll ,2,3,4-tetrazole-5-thiomethyl-A cephem-4-carboxylic acid: The above preparation was repeated using l-methyl-1,2,3,4-tetrazole-5-thiol instead of the thiadiazole. There was obtained 25 g. (76 percent) of 7-amino-3-(1-methyl-1,2,3,4-tetrazole-5 thiomethyl)- A -cephem-4-carboxylic acid. The preparation of this same compound is also described in US. Pat. 3,516,997 in column 6 under the heading Preparation 7.

CHzCOzH 2,4-DNP D00 CHaNH-B 00 m lamin 1 5 EXAMPLE A 7- (o-aminomethylphenylacetarnido) -3-[ 1-methyl-1,2,3,4,- tetrazol-S-thio) methyl] -ceph-3 -em-4-carboxylic acid N,N'-dicyclohexycar.bodiimide (1.44 g., 0.0070 mole) was added in one portion to an ice-cooled solution of -tert butoxycarbonylaminomethylphenylacetic acid (1.86 g., 0.0070 mole) and 2,4-dinitrophenol (1.29 g., 0.0070 mole) in 15 ml. of ethyl acetate. The mixture was kept at 0 for 30 min., then at 25 for an additional 30 min. The precipitate was removed by a filtration and the solvent removed from the filtrate. To the crude activated ester was added, at 0, a solution of 7-amino-3-[(1- methyl-1,2,3,4-tetrazol--thio)methy11ceph-3-em 4 carboxylic acid (2.30 g., 0.0070 mole) and triethylamine (1.41 g., 0.014 mole) in 15 ml. of methylene chloride. The reaction mixture was stirred at room temperature for 2 hours, a small amount of insoluble material was removed by filtration and the filtrate diluted with ether. The oily precipitate was twice redissolved in a small amount of methylene chloride and reprecipitated with ether. It was then dissolved in a small amount of methanol and this solution treated with 3.5 ml. of a 2.3 M solution of potassium Z-ethylhexanoate in n-butyl alcohol. Ether was added and the precipitated potassium salt of the protected cephalosporin (2.2 g.) was collected by filtration. An aqueous solution of the potassium salt was layered with ethyl acetate and, with stirring, acidified to pH 2.5. The ethyl acetate layer was dried and concentrated to give the cephalosporanic acid as a foam (1.3 g.). This was treated, at 0, for one hour with 10 ml. of trifluoroacetic acid followed by the addition of ether. The precipitate was collected, washed with ether, and dissolved in a mixture of water (20 ml.) and acetone (20 ml.). The pH was adjusted to 5.0 with dilute ammonium hydroxide. Then the solution was concentrated under reduced pressure to a small volume and cooled. The product precipitated and was collected by filtration and washed with icewater, methanol and ether respectively, yield 0.45 g. (14% The infrared and n.m.r. spectra fully agreed with the assigned structure; the purity was estimated at 85%.

EXAMPLE B 7-(o-aminomethylphenylacetamido) -3-[ (5-methyl-1,3,4- thiadiazolylthio) methyl] ceph-3-em-4-carboxylic acid The cephalosporin was prepared by the same procedure given in detail in Example A but using 7-amino-3-[(5- methyl-1,3,4-thiadiazolylthio)methyl] ceph-3-em 4 carboxylic acid. The purity of the product obtained was estimated at 90%; infrared maxima (Nujol mull): 3300 (NH), 1765 (fl-lactam carbonyl and 1660 cm.- (amidocarbonyl EXAMPLE C 7- (o-aminomethylphenylacetamido) -3- [S 1,2,3-triazole- 5-yl)thiomethyl]-ceph-3-em-4-carboxylic acid (M.I.C.) in meg/ml. versus the indicated microorganisms as determined, by overnight incubation at 37 C. by Tube Dilution. Results with cephalothin are also given.

TABLE [M.I.C. in meg/ml] Expert. No. 1 Expert. No. 2

New New cpd. Cephacpd. Cepha- Organism No. 1 lothin No. 2 lothin D. pneumoniae plus A9585 01 .04 005 04 5% serum. .01 08 005 08 Str. pyogenes plus A9604 01 04 01 04 5% serum. 01 08 01 08 S. aureus Smith A9537 .16 .08 V .08 .16 16 16 04 16 S. aureus Smith plus A9537 16 3 16 3 serum. 16 .3 16 3 S. aureus BX1633-2 A9606 16 16 16 I 3 at 10- diln. V 16 16 16 3 S. aureus BL1633-2 A9606 5 3 5 3 at 10- diln. .3 .6 .5 .6

S. aureus meth.- A15097 2 1 4 2 resistant. 5 2 1 1 Sal. enterilidis A9531 25 16 25 3 08 3 08 3 E. coli .Tuhl A15119 v 25 16 .5 16 3 16 5 8 E. coli A9675 4 y as 2 ea 1 63 2 63 K. pneumoniae A9977 25 1 5 2 16 2 5 1 K. pneumoniae A15130 1 16 1 16 1 32 1 16 Pr. mimbilis A9900 5 1 .5 1 25 1 .5 1

Pr. morgum'i A15153 4 4 125 4 125 4 125 Paeruginosa A9843A 125 125 125 125 125 125 125 125 Ser. 'marcescens A20019 63 125 63 125 16 125 32 125 "50% nutrient broth, 45% antibiotic assay broth.

7 (o aminomethylphenylacetamido)-3-[(l-methyl-S- tetrazolylthio)methyl-ceph-3em-4-carboxylic acid and 7- (o-aminomethylphenylacetamido)-3-[(5 methyl 1,3,4- thiadiazolylthio)methyl]ceph-3-em-4-carboxylic acid were well absorbed in mice upon parenteral, but not oral, administration. A lower minimum dose (CD of each of these two compounds than of cephalothin was required by subcutaneous administration in two doses to cure 50% of groups of mice infected with E. coli Juhl (A15119). Example A, 7 (0 aminornethylphenylacetamido)-3-[(1- methyl-1,2,3,4-tetrazol-5-thio)methyl]-ceph-3-em 4 carboxylic acid is also named 7-(o-aminomethylphenylacetamido) 3 [(1-methyl-5-tetrazolylthio)methyl]-ceph-3- em-4-carboxylic acid. I

We claim:

1. A compound of the formula cmmcmsoma). I 

1. A COMPOUND OF THE FORMULA 